Letter reSeArCH
Extended Data Fig. 3 | Inducible overexpression of FOXA1 variants
influences organoid lumen size and morphology. a, Schematic of
doxycycline-inducible pCW-FOXA1 constructs used in the study.
b, W estern blot analysis of lysates from pCW-FOXA1(WT) organoids
following acute doxycycline treatment. Representative blot, experiment
repeated two independent times with similar results. For source gel
data, see Supplementary Fig. 1. c, Western blot analysis of lysates from
organoids following long-term doxycycline treatment. Sizes of endogenous
and Flag-tagged FOXA1 are noted, as well as the smaller truncated form
from G275X at the expected size ~38 kDa. Representative blot, experiment
repeated three independent times with similar results. For source gel data,
see Supplementary Fig. 1. d, Quantification of lumen areas measured at
ten days after seeding. Solid black bar represents geometric mean. Values
for sample size (indicated as dots) and P values are as follows: EV (292),
+WT (284, P < 0.0001 over EV), +R219S (60, <0.0001), +F254_E255del
(119, <0.0001), +D226N (120, <0.0001), +R261C (114, <0.0001),
+R219C (333, 0.2915), +G275X (75, <0.0001), +M253_N256del
(150, 0.2006), +M253K (63, 0.2343), +Y259S (32, 0.2045), +Y259C
(45, 0.0082), +F266L (107, 0.1219), +H247Q (63, 0.8343), +H247R
(180, <0.0001), +H247Y (71, 0.9104). All P values are relative to WT
unless noted, calculated using unpaired, two-tailed Student’s t-test.
Colours represent location of mutation within FOXA1. e, Histology and
immunohistochemistry of organoid lines overexpressing additional alleles
of FOXA1 via the doxycycline-inducible pCW vector 10 days after seeding.
Images from a single biological experiment.